The primary interest of this laboratory is analysis of mammalian fertilization at the molecular level, combining techniques of cell biology, biochemistry and molecular genetics. This laboratory has identified a mouse sperm protein, a lectin called sp56, which has many properties expected of the protein responsible for sperm recognition of the mouse egg's zona pellucida (ZP). sp56 (Mr 56,000 daltons) has specific affinity for ZP3, the egg's receptor for sperm, and for ZP3's receptor oligosaccharide, the part of ZP3 recognized by sperm. sp56 is a homomultimeric, extracellular, peripheral membrane protein, restricted to the plasma membrane overlying the sperm head, where ZP3 recognition sites are located. sp56 is apparently anchored to the sperm plasma membrane by another protein, called PC. Immunological and molecular probes have revealed that sp56 is expressed exclusively in spermatogenic cells of the mouse, and that its presence or absence on sperm of other mammals can account for species-specificity of sperm-egg recognition in mammals. The long-term goals addressed by studies described here are 1) to prove or disprove the hypothesis that sp56 is the sperm protein which recognizes ZP3 in the mouse egg ZP, mediating sperm-egg binding, 2) to determine the molecular structure of the sp56/ZP3 complex, and 3) to characterize the biochemical pathway leading from sperm-egg recognition to the acrosome reaction in mouse. Future studies will focus on identification of analogous human sperm proteins. Identification and characterization of these proteins will undoubtedly lead to specific clinical methods for perturbing fertilization and for detecting and, perhaps, bypassing defects in fertilization. Experiments proposed here are designed to test the hypothesis that sp56 associates with ZP3 during the normal course of fertilization, thereby mediating binding of the sperm head to the ZP. Complementary studies are designed to characterize sp56 expression and to determine whether, through its attachment to the sperm plasma membrane, sp56 recognition of ZP3 triggers the acrosome reaction. Specific Aim #1 To analyze recognition between sp56 and ZP3. Using immunohistochemical methods, studies will be designed to determine whether sp56 is identical to ZP3 binding sites on the sperm surface. Affinity studies will be performed to determine whether sp56, like sperm, discriminates between ZP3 and ZP3f. Specific Aim #2 To determine whether transcription of the sp56 gene occurs first in haploid spermatids. Nucleic acid probes will be used to detect newly expressed sp56 mRNA in spermatogenic cells. Specific Aim #3 To identify the carbohydrate recognition domain (CRD) of sp56. In vitro mutagenesis of sp56, expressed in a heterologous cell line, will be used to determine those domains of the protein necessary for recognition of ZP3's FD oligo. Specific Aim #4 To characterize the newly identified mouse sperm protein, PC, which apparently anchors sp56 to the sperm surface. Patch- clamp studies of PC, integrated into lipid bilayers, will be used to determine whether this protein is an ion channel and, therefore, responsible for triggering calcium uptake and membrane fusion in the sperm. Antibodies raised against the protein will be used to test whether 'patching' of PC directly triggers the acrosome reaction and to determine whether PC is a ubiquitous lectin-anchoring protein on sperm of other species.